Characterization of Novel Klebsiella Phage PG14 and Its Antibiofilm Efficacy

ABSTRACT The increasing frequency of infections caused by multidrug-resistant Klebsiella pneumoniae demands the development of unconventional therapies. Here, we isolated, characterized, and sequenced a Klebsiella phage PG14 that infects and lyses carbapenem-resistant K. pneumoniae G14. Phage PG14 showed morphology similar to the phages belonging to the family Siphoviridae. The adsorption curve of phage PG14 showed more than 90% adsorption of phages on a host within 12 min. A latent period of 20 min and a burst size of 47 was observed in the one step growth curve. Phage PG14 is stable at a temperature below 30°C and in the pH range of 6 to 8. The PG14 genome showed no putative genes associated with virulence and antibiotic resistance. Additionally, it has shown lysis against 6 out of 13 isolates tested, suggesting the suitability of this phage for therapeutic applications. Phage PG14 showed more than a 7-log cycle reduction in K. pneumoniae planktonic cells after 24 h of treatment at a multiplicity of infection (MOI) of 10. The phage PG14 showed a significant inhibition and disruption of biofilm produced by K. pneumoniae G14. The promising results of this study nominate phage PG14 as a potential candidate for phage therapy. IMPORTANCE Klebsiella pneumoniae is one of the members of the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group of pathogens and is responsible for nosocomial infections. The global increase of carbapenem-resistant K. pneumoniae has developed a substantial clinical threat because of the dearth of therapeutic choices available. K. pneumoniae is one of the commonly found bacteria responsible for biofilm-related infections. Due to the inherent tolerance of biofilms to antibiotics, there is a growing need to develop alternative strategies to control biofilm-associated infections. This study characterized a novel bacteriophage PG14, which can inhibit and disrupt the K. pneumoniae biofilm. The genome of phage PG14 does not show any putative genes related to antimicrobial resistance or virulence, making it a potential candidate for phage therapy. This study displays the possibility of treating infections caused by multidrug-resistant (MDR) isolates of K. pneumoniae using phage PG14 alone or combined with other therapeutic agents.

Results section headings are generally not descriptive of the text, especially first two -I suggest rewriting them to emphasize the specific results. In addition, some results sections (body text) lack detail (one is only one sentence long).
Most figure legends need more detail about what was done. Preferably the authors should get help with figure legend and figure conventions, and with stats.
Figures are almost all blurry/hard to read, need work getting to publication quality.

Examples of issues with figures and figure legends:
-Aa / Ab / Bb naming in figs is confusing Table 1 -EOP usually a quantitative value: what does each qualitative result correspond to? Should appear in legend. Would actually suggest a quantitative assessment if reporting as EOP Host range: it would be nice to know why the strains chosen for host range were chosen (both in the text and in table 1). Section: 'Antibiofilm activity of phage PG14' -'polysaccharide was its major component'. What kind of polysaccharide? how was this determined? -very sparse section, need details on biochemical characterization mentioned Discussion -Very long run-on paragraphs -check wording: 'to the suite of the basis for the assessment' Writing: The writing has several issues with sentence structure, punctuation, word choice, capitalization, inconsistencies, etc Examples: -switch from S to + -check comma usage throughout -check definition of opaque -define acronyms TEM, EOP, SEM and others the first time they are used -colloquial language 'getting adsorbed' -imprecise language in places 'which was possibly estimated in crystal violet assay' -what does 'possibly estimated' mean? -suggest reconsidering use of the word 'futuristic' (re: enzybiotics) This paper focuses on the characterisation of phage PG14 that infects multidrug resistant Klebsiella pneumoniae and has potential for phage therapy. This subject area is vitally important from a global health perspective as carbapenem-resistant K. pneumoniae is an increasing clinical threat with few antimicrobial treatments available, phages could be increasingly important for therapy. This is a through and well written account of the isolation of a phage and its ability to degrade biofilms for future therapeutic applications.

Specific points
Try not to switch between CRKP and K. pneumoniae, stick to CR K. pneumoniae if you want to make the difference clear, but it is confusing to have CRKP and CLSM in the text as they are unfamiliar accronyms.
In "Importance" Line 25: I am not sure the statement "Biofilms responsible for device-related infections are mostly caused by K. pneumoniae." Is true, it is not backed up by references in the introduction.
Ensure all statements have references, e.g. Lines 41-2 add reference for "In addition, to its alarming antibiotic-resistant nature, K. pneumoniae displays a high degree of virulence enabling it to invade and survive in the host." Lines 63-4 "several reports of phages ..." but the sentences only has one reference, add more to back up "several".
The text in all figures is too small, make text on figures and graph axes bigger. Line 292 change "futuristic" to "future" Line 299 states "CV staining assay, TVC assay and confocal microscopic analysis have confirmed the ability of phage PG14 to significantly inhibit and disrupt the biofilm" the results show that CV showed no biofilm degradation (Fig 5 A). Change the text.

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Reviewer comments:
Reviewer #1 (Comments for the Author): Summary: The manuscript contains useful data and represents a thorough characterization of the phage. It also has a nicely detailed methods section. However, the presentation of results sections (figures, figure legends, results) needs work.

Main issues:
Results section headings are generally not descriptive of the text, especially first two -I suggest rewriting them to emphasize the specific results. In addition, some results sections (body text) lack detail (one is only one sentence long).      The main focus of our study was to isolate and characterize the phages against Klebsiella pneumoniae and to study their antibiofilm activity. Therefore, only basic characterization of the biofilm matrix was carried out in the present study.

Discussion
-Very long run-on paragraphs -check wording: 'to the suite of the basis for the assessment' Reply: Paragraphs are modified as per the suggestion. Refer to lines 178 to 179, 193 to 196, 200 to 202, 205-206 and 217 to 221.

Writing:
The writing has several issues with sentence structure, punctuation, word choice, capitalization, This paper focuses on the characterization of phage PG14 that infects multidrug resistant Klebsiella pneumoniae and has potential for phage therapy. This subject area is vitally important from a global health perspective as carbapenem-resistant K. pneumoniae is an increasing clinical threat with few antimicrobial treatments available, phages could be increasingly important for therapy. This is a through and well written account of the isolation of a phage and its ability to degrade biofilms for future therapeutic applications.

Specific points
Try not to switch between CRKP and K. pneumoniae, stick to CR K. pneumoniae if you want to make the difference clear, but it is confusing to have CRKP and CLSM in the text as they are unfamiliar accronyms.
Reply: Modified the statement as per the suggestion In "Importance" Line 25: I am not sure the statement "Biofilms responsible for device-related infections are mostly caused by K. pneumoniae." Is true, it is not backed up by references in the introduction.
Reply: Modified the statement as per the suggestion and references are added. Refer lines 44 to 46 Ensure all statements have references, e.g. Lines 41-2 add reference for "In addition, to its alarming antibiotic-resistant nature, K. pneumoniae displays a high degree of virulence enabling